The present invention relates to devices and methods for detecting secreted cellular products, and in particular for measuring secreted T cell products, including cytokines.
Over the last thirty years, the immune system has been studied with better and better laboratory tools. Still, most knowledge of the immune response concerns antibody formation. This is understandable given that antibodies, including specific antibodies, are easily detectable in quantity in the serum of immunized individuals. Antibodies are products of B lymphocytes. Antibody production by individual B cells (as well as cells fused with B cells such as hybridomas) is also readily achieved in vitro using a variety of tests, including the ELISA spot assay (also called xe2x80x9cELISPOTxe2x80x9d for xe2x80x9cenzyme-linked immunospotxe2x80x9d). See Segwick, J. D. and Holt, P. G., xe2x80x9cA solid-Phase Immunoenzymatic Technique for the Enumeration of Specific Antibody-Secreting Cells,xe2x80x9d J. Immunol. Methods 57:301-309 (1983). See also Mazer, B. D. et al., xe2x80x9cAn ELISA Spot Assay for Quantitation of Human Immunoglobulin Secreting Cells,xe2x80x9d J. Allergy Clin. Immunol. 88:235-243 (1991).
In the conventional B cell ELISA spot assay, standard, commercially-available flat-bottom plates (containing no membranes) are coated overnight with antigen or animal antibody. In the case where antibody is used, it is typically an xe2x80x9canti-antibodyxe2x80x9d (e.g., goat antibody reactive with human IgG, IgE, IgM etc.). After blocking overnight, B cells are introduced in the wells. Following a sufficient culture period, the wells are washed free of the cells and an antibody-enzyme conjugate is added. The plates arc then developed using substrate for the enzyme of the conjugate. Spots are counted using a microscope. The lowest amount of detectable antibody is typically in the range of 10 to 50 picograms. See e.g., Renz, H. et al., xe2x80x9cEnhancement of IgE Production by Anti-CD40 Antibody in Atopic Dermatitis,xe2x80x9d J. Allergy Clin. Immunol. 93:658-668 (1994).
In contrast to the antibody response, the response of T cells to antigen (including the antigen of pathogens) can not be easily monitored due to the fact that antigen reactive T cells occur in low frequencies and the fact that their secretory products are not typically stable (i.e. have a short half-life). Indeed, even in hyper-immunized individuals, antigen reactive T cells constitute 1 in 10,000 cells or less in the peripheral T cell pool, e.g., among the T cells in circulating blood. Thus, T cells usually act beyond the detection limits of conventional assay systems (such as proliferation assays).
As a consequence of this, there is no technique at present available that would reliably measure whether a patient has generated a T cell response to a particular pathogen, such as HIV. There is no reliable assay that can detect whether a T cell response to HIV proteins has been generated, what proteins of the virus are primarily targeted, and which determinants within that protein are immunodominant. There is also no reliable method available for testing the magnitude of the anti-viral T cell response (clonal sizes) and its quality (e.g. whether the response is pro- or anti-inflammatory).
The heterogeneity of T cells, their products and the mode of function provide great challenges (particularly as compared to B cells). With respect to mode of function, T cells can act in different subpopulations that utilize strikingly different effector functions. T cell responses can be pro-inflammatory T helper 1 type, Th1, characterized by the secretion of interferon gamma (IFNxcex3) and interleukin 2 (IL-2). Th1 cells are critical for the cellular defense and provide little help for antibody secretion. (Strong Th1 responses are usually associated with poor antibody production, which highlights the importance of directly measuring the T cell response instead of relying on antibody measurements.) The other class of T cell responses is anti-inflammatory, mediated by Th2 cells that produce IL-4, 5, 10, but no IL-2 or IFNxcex3. Th2 cells are the helper cells for antibody production. CD4+ and CD8xe2x88x92 cells both occur in these subpopulations: Th1/Th2:CD4, TC1/TC2:CD8.
Importantly, for each type of infection there is an xe2x80x9cappropriatexe2x80x9d (and different) type of T cell response (e.g., Th1 vs. Th2, CD4+ vs. CD8+) that clears the infectious agent but does not cause excessive tissue destruction to the host. It is detrimental to the host if an xe2x80x9cinappropriatexe2x80x9d type of T cell response is engaged (Th1 instead of Th2, or vice versa). Thus, there is a strong need for assessing the host""s T cell immunity to the virus to understand the host-virus interplay and to design vaccines. An ideal assay should permit monitoring all of the critical features of the T cell response: first, the existence of a response, i.e., that effector cells have been generated, second the nature of the effector cells as Th1 or Th2 type cells, and finally the magnitude of the response.
Some attempts have been made to apply the B-cell ELISA spot technology to T cells. However, the conventional cytokine ELISA spot assay has not been a more sensitive tool than alternative assays (e.g. proliferation assays), displaying high background and generally a weak signal. The conventional ELISA spot assay for T cells involves plates containing nitrocellulose membranes which are precoated with a capture antibody specific for the cytokine to be detected. See e.g., Taguchi, T. et al., xe2x80x9cDetection of Individual Mouse Splenic T Cells Producing IFNxcex3 and IL-5 Using the Enzyme-Linked Immunospot (ELISPOT) Assay,xe2x80x9d J. Immunol. Methods 128:65-73 (1990). See also Fujihashi, F. et al., xe2x80x9cCytokine-Specific ELISPOT Assay,xe2x80x9d J. Immunol. Methods 160:181-189 (1993). See also Miyahira, Y. et al., xe2x80x9cQuantification of Antigen Specific CD8xe2x88x92 T Cells Using an ELISPOT Assay,xe2x80x9d J. Immunol. Methods 181:45-54 (1995). T cells are plated with the test antigen and start to secrete the type of cytokine they are programmed to produce. As the cytokine is released, it is captured around the secreting cells by the plate bound antibody. After 24 h the cell culture is terminated, cells are removed and the plate bound cytokine is visualized by a second antibody and an enzymatic color reaction.
Ideally, each cytokine producing cell will be represented as an ELISA spot. However, with conventional assays, sensitivity does not exceed cytokine measurements in the supernatant by ELISA (cytokine measurements in culture supernatants provide a positive result only if more than 1000 cells are present per well). The quantification of the data is also problematic because of background problems and the subjective, visual evaluation.
There is a great need for better assays to measure secreted T cell cytokines. Specifically, there is a need for devices and methods with greater capability to detect cytokines from individual cells in a mixture of heterogeneous cells.
The present invention relates to devices and methods for detecting cellular products, and in particular for measuring secreted T cell products, including cytokines. The present invention contemplates a device and method comprising a membrane. In one embodiment, the membranes of the present invention are weakly hydrophilic and display advancing contact angles for water between approximately seventy (70) and approximately ninety (90) degrees. In another embodiment, the membranes of the present invention are hydrophobic and display advancing contact angles for water between approximately ninety (90) and approximately one hundred and thirty (130) degrees. In another embodiment, the membranes of the present invention are very hydrophobic and display advancing contact angles for water greater than approximately one hundred and thirty (130) degrees.
In accordance with one embodiment of the invention, the microwells containing the hydrophobic membrane of the present invention are precoated with a first capture reagent (see FIG. 1A). It is not intended that the present invention be limited by the nature of the capture reagent. In one embodiment, the capture reagent is a cytokine binding ligand such as a capture antibody specific for the cytokine to be detected (e.g., anti-IFNxcex3 mAb1 or anti-IL5 mAb or with both simultaneously for the two color assay). In a preferred method, freshly isolated, primary cell populations (e.g., lymph node, spleen cells, etc.) are subsequently plated (see FIG. 1B) with the test antigen, e.g., HIV protein or peptide; control cultures contain irrelevant antigens or peptides. Since the primary cell suspensions contain abundant antigen presenting cells (APC) to process and present the antigen, specific T cells become activated and start to secrete the type of cytokine they are programmed to produce. As the cytokine is released, it is captured around the secreting cells by the plate-bound capture reagent (see FIG. 1B). After a suitable culture period (e.g., between approximately 30 minutes and 48 hours), the cell culture is terminated and the cells are removed (e.g. by washing), leaving the captured, plate-bound secreted product (see FIG. 1C).
The plate-bound, captured cytokine is visualized by a detection reagent. It is not intended that the present invention be limited by the nature of the detection reagent. In one embodiment, the detection reagent is a second cytokine binding ligand (e.g., antibody) free in solution that is conjugated to enzyme (see FIG. 1D). In another embodiment, the present invention contemplates the use of directly labelled detection reagents (e.g. antibodies). The addition of substrate (see FIG. 1E) results in an enzymatic color reaction. Each cytokine producing cell will be represented as an ELISA spot.
In still another embodiment, the detection reagents is directly labelled with a fluorochrome (e.g., FITC, PE or texas red) or with colored beads (different colors for different secretory products) or a ligand like biotin that can be detected with tertiary reagent that is labelled as above (with a fluorochrome, bead or enzyme).
While one embodiment of the present invention employs, as a first capture reagent, cytokine binding antibodies bound to a hydrophobic membrane to capture cytokines, the present invention also contemplates the binding of soluble products directly to the membrane without the use of capture reagents. In still another embodiment, the present invention contemplates non-antibody ligands, such as cytokine receptors and lectins (e.g. concanavalin A), as the capture ligand.
The present invention contemplates a testing device comprising: a plurality of microwells, a membrane within each of said microwells, wherein said membrane displays advancing contact angles for water greater than approximately seventy degrees, and a first cytokine binding ligand bound to said membrane. In one embodiment, said membrane displays advancing contact angles for water between approximately ninety (90) and approximately one hundred and thirty (130) degrees. In another embodiment, said membrane displays advancing contact angles for water greater than approximately one hundred and thirty (130) degrees. In a preferred embodiment, said membrane is a hydrophobic, PVDF-based membrane or a hydrophobic, nylon-based membrane.
The present invention contemplates a variety of first cytokine binding ligands, including antibody specific for a cytokine (e.g. antibody specific for an interferon such as interferon gamma or antibody specific for an interleukin, such as IL-5) as well as cytokine receptors (e.g. interleukin receptors). Where more than one cell product is to be detected, the present invention contemplates a second cytokine binding ligand bound to said membrane.
In a specific embodiment, the present invention contemplates, a method of detecting secreted T cell cytokines, comprising: a) providing: i) a microwell comprising a hydrophobic membrane having a first cytokine binding ligand; ii) a primary cell population comprising T cells capable of secreting cytokines; b) adding said primary cell population to said microwell under conditions such that said T cell secretes a cytokine that binds to said first cytokine binding ligand; and c) detecting said secreted T cell cytokine. The present invention further contemplates an embodiment wherein said microwell further comprises, prior to said adding of step (b), a test antigen (such as a peptide).
The present invention is not limited by the nature of the primary cell population In one embodiment, the primary cell population further comprises antigen presenting cells.
The present invention contemplates a variety of detecting schemes. In one embodiment, said detecting comprises introducing a second cytokine binding ligand into said microwell under conditions such that a colorimetric signal is generated.
The present invention contemplates computer image analysis. In one embodiment, the analysis method detects and categorizes spots on a membrane, and comprises: a ) providing a testing device, comprising i) a plurality of microwells, and ii) a membrane (such as a hydrophobic membrane) within each of said microwells, wherein said membrane has been treated under conditions such that spots could develop; b) capturing an image of each of said membranes from each of said microwells; c) digitizing said images to create digitized images comprising bits; and d) thresholding said digitized images, such that said spots on said membranes are detected and categorized. Said thresholding may comprise superimposing an inverted binary bit map of a predetermined threshold on each of said digitized images and eliminating those bits not meeting the threshold.